The rate-determining step on the recA protein-catalyzed ssDNA-dependent ATP hydrolysis reaction pathway.

We recently constructed a mutant recA protein in which His 163 was replaced by a tryptophan residue. The [H163W]recA protein is functionally identical to the wild-type protein, and the Trp163 side chain serves as a fluorescence reporter group for the ATP and ATPgammaS-mediated conformational transitions of the [H163W]recA-ssDNA complex. In this report, the pre-steady-state kinetics of the ATP and ATPgammaS-mediated transitions were examined by stopped-flow fluorescence. The kinetics of the ATP-mediated fluorescence change were consistent with a two-step mechanism in which an initial rapid equilibrium binding of ATP to the recA-ssDNA complex is followed by a first-order isomerization of the complex to a new conformational state; the rate constant for the isomerization step of 18 min is identical to the steady-state turnover number for ATP hydrolysis. The kinetics of the ATPgammaS-mediated fluorescence change were also consistent with a two-step binding mechanism with a unimolecular isomerization of 18 min(-1); since ATPgammaS is not hydrolyzed appreciably on the time scale of these experiments (0.017 min(-1)), this indicates that the isomerization step follows ATPgammaS (or ATP) binding but precedes ATPgammaS (or ATP) hydrolysis. These and other results are consistent with a kinetic model in which an ATP-mediated isomerization of the recA-ssDNA complex is the rate-determining step on the recA protein-catalyzed ssDNA-dependent ATP hydrolysis reaction pathway.

Reengineering the nucleotide cofactor specificity of the RecA protein by mutation of aspartic acid 100.

We have recently obtained evidence for a direct linkage between the S0.5 (S0.5 is the substrate concentration required for half-maximal velocity) value of a nucleoside triphosphate and the conformational state of the RecA-ssDNA complex, with an S0.5 value of 125 microM or less required for stabilization of the strand exchange-active conformation. For example, although ATP and ITP are hydrolyzed by the RecA protein with the same turnover number (18 min-1), ATP (S0.5 = 45 microM) functions as a cofactor for the strand exchange reaction, whereas ITP (S0.5 = 500 microM) is inactive as a strand exchange cofactor. The RecA protein crystal structure suggests that cofactor specificity is determined by Asp100, which likely forms a hydrogen bond with the exocyclic 6-amino group of ATP; the higher S0. 5 value for ITP is presumably due to unfavorable interactions between Asp100 and the 6-carbonyl group of the inosine ring. To test this hypothesis, we prepared a mutant RecA protein in which Asp100 was replaced by an asparagine residue. The S0.5(ITP) for the [D100N]RecA protein is 125 microM, indicating favorable interactions between the Asn100 side chain and the 6-carbonyl group of ITP. Correspondingly, ITP functions as a cofactor for the strand exchange activity of the [D100N]RecA protein. This result demonstrates the importance of the residue at position 100 in determining nucleotide cofactor specificity and underscores the importance of the S0.5 value in the RecA protein-promoted strand exchange reaction.

Spectroscopic demonstration of a linkage between the kinetics of NTP hydrolysis and the conformational state of the recA-single-stranded DNA complex.

We recently constructed a mutant recA protein in which His-163 was replaced by a tryptophan residue; the [H163W]recA protein is functionally identical to the wild-type protein, and the Trp-163 side chain serves as a reporter group for the conformational transitions of the [H163W]recA-single-stranded DNA (ssDNA) complex. We have now examined the fluorescence properties of the [H163W]recA-ssDNA complex in the presence of a series of alternate nucleoside triphosphate cofactors. Under standard conditions (pH 7.5), ATP (S0.5 = 70 microM) and purine riboside triphosphate (PTP) (S0.5 = 110 microM) effect a 44% decrease in Trp-163 fluorescence and are active as cofactors for the DNA strand exchange reaction. In contrast, ITP (S0.5 = 400 microM) elicits only a 20% decrease in Trp-163 fluorescence (a level identical to that observed with the nucleoside diphosphates ADP, PDP, and IDP) and is inactive as a strand exchange cofactor. If the S0.5 (PTP) is increased to 130 microM (by increasing the pH of the reaction solution), the PTP-mediated quenching of Trp-163 fluorescence decreases to 20%, and PTP becomes inactive as a strand exchange cofactor. These results provide direct evidence for a linkage between the S0.5 value of a nucleoside triphosphate and the conformational state of the recA-ssDNA complex, with an S0.5 of 100-120 microM or lower required for stabilization of the strand exchange-active conformation.

Interaction of gamma-glutamyl transpeptidase with acivicin.

Inactivation of gamma-glutamyl transpeptidase by acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid) is rapid, thought to be irreversible, and associated with binding of close to 1 mol of inhibitor/mol of enzyme. Previous studies with [3-14C]acivicin indicated binding (prevented by substrate) to a specific hydroxyl group (threonine 523) of the rat kidney enzyme. In the present work, we found that such inactivation can be reversed by treating the inhibited enzyme with hydroxylamine. Reactivation (more than 85% complete) is associated with release from the inactivated enzyme of compounds that exhibit the properties of threo-beta-hydroxy-L-gamma-glutamyl hydroxamate and 3-hydroxypyrrolidone-2-carboxylate. We found that the enzyme acts very slowly on acivicin, at a rate that is about 10(-9) that of its normal catalytic rate with glutathione, to form threo-beta-hydroxy-L-glutamate and hydroxylamine. The findings indicate that inhibition by acivicin involves its transformation on the enzyme to an inhibitory species which is attached, apparently by ester linkage, to a specific hydroxyl group of the enzyme. The very slow rate of release of this intermediate appears to account for the observed inhibition.

Introduction of a tryptophan reporter group into loop 1 of the recA protein. Examination of the conformational states of the recA-ssDNA complex by fluorescence spectroscopy.

Site-directed mutagenesis was used to replace His-163 in the Loop 1 region of the recA protein with a tryptophan residue. The [H163W]recA protein binds single-stranded DNA (ssDNA), catalyzes ssDNA-dependent ATP hydrolysis, and is fully active in the three-strand exchange reaction. In addition, the fluorescence properties of the Trp-163 reporter group are very sensitive to the binding of nucleotide cofactors to the H163W]recA-ssDNA complex. The fluorescence of Trp-163 is modestly quenched by the binding of ADP (21%) and strongly quenched by the nonhydrolyzable ATP analog, ATP gamma S (70%); since ADP and ATP gamma S stabilize the closed and open conformations of the recA-ssDNA complex, respectively, the quenched states observed with these nucleotides likely reflect differences in the fluorescence properties of tryptophan 163 in these two states. ATP has a more complex time-dependent effect on Trp-163 fluorescence. When ATP is added to [H163W]recA-ssDNA complexes, there is an immediate quenching of Trp-163 fluorescence (44%) which is intermediate in intensity between that observed with ADP and ATP gamma S. The ATP-induced quenching gradually decreases with time as the pool of ATP is converted to ADP by the ATP hydrolysis activity of the [H163W]recA protein. These results are discussed with regard to the nucleotide cofactor-dependent conformational transitions of the recA-ssDNA complex.

Inhibition of glutathione synthesis in the newborn rat: a model for endogenously produced oxidative stress.

A model for oxidative stress is described in which glutathione (GSH) synthesis is selectively blocked in newborn rats by administration of L-buthionine-(S,R)-sulfoximine (BSO). In this model, the normal endogenous physiological formation of reactive oxygen species is largely unopposed, and therefore oxidative tissue damage occurs; because GSH is used for reduction of dehydroascorbate, tissue ascorbate levels decrease. In lung there are decreased numbers of lamellar bodies and decrease of intraalveolar surfactant. Proximal renal tubular, hepatic, and brain damage also occur. A diastereoisomer of BSO that does not inhibit GSH synthesis, L-buthionine-R-sulfoximine, does not produce toxicity; this control experiment renders it unlikely that the observed effects of BSO are produced by the sulfoximine moiety itself. There is correlation between the decrease of mitochondrial GSH levels and mitochondrial and cell damage. Oxidative stress as evaluated by mitochondrial damage and mortality can be prevented by treatment with GSH esters or ascorbate. There is apparent linkage between the antioxidant actions of GSH and ascorbate. This model, which may readily be applied to evaluation of the efficacy of other compounds in preventing oxidative stress, offers an approach to study of other effects of GSH deficiency (e.g., on lipid metabolism, hematopoiesis), and closely resembles oxidative stress that occurs in certain human newborns and in other clinical states.

Interaction of gamma-glutamyl transpeptidase with glutathione involves specific arginine and lysine residues of the heavy subunit.

Gamma-glutamyl transpeptidase, an enzyme of importance in glutathione metabolism, consists of two subunits, one of which (the light subunit, Mr 22,000; residues 380-568; rat kidney) contains residue Thr-523, which selectively interacts with the substrate analog acivicin to form an adduct that is apparently analogous to the gamma-glutamyl enzyme intermediate formed in the normal reaction (Stole, E., Seddon, A. P., Wellner, D., and Meister, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1706-1709). The present studies indicate that specific arginine and lysine residues of the heavy subunit (Mr 51,000; residues 31-379) participate in catalysis by binding the substrates. Selective labeling studies of the enzyme with [14C]phenylglyoxal showed that Lys-99 and Arg-111 were modified. This appears to be the first instance in which phenylglyoxal was found to react with an enzyme lysine residue. Incorporation of [14C]phenylglyoxal into Lys-99 was decreased in the presence of acceptor site selective compounds. Incorporation into both Lys-99 and Arg-111 was decreased in the presence of glutathione. The findings suggest that Lys-99 and Arg-111 interact, respectively, with the omega- and alpha-carboxyl groups of glutathione. That these putative electrostatic binding sites are on the heavy subunit indicates that both subunits contribute to the active center. Two additional heavy subunit arginine residues become accessible to modification by phenylglyoxal when acivicin is bound, suggesting that interaction with acivicin is associated with a conformational change.

Glutathione deficiency leads to mitochondrial damage in brain.

Glutathione deficiency induced in newborn rats by giving buthionine sulfoximine, a selective inhibitor of gamma-glutamylcysteine synthetase, led to markedly decreased cerebral cortex glutathione levels and striking enlargement and degeneration of the mitochondria. These effects were prevented by giving glutathione monoethyl ester, which relieved the glutathione deficiency, but such effects were not prevented by giving glutathione, indicating that glutathione is not appreciably taken up by the cerebral cortex. Some of the oxygen used by mitochondria is known to be converted to hydrogen peroxide. We suggest that in glutathione deficiency, hydrogen peroxide accumulates and damages mitochondria. Glutathione, thus, has an essential function in mitochondria under normal physiological conditions. Observations on turnover and utilization of brain glutathione in newborn, preweaning, and adult rats show that (i) some glutathione turns over rapidly (t 1/2, approximately 30 min in adults, approximately 8 min in newborns), (ii) several pools of glutathione probably exist, and (iii) brain utilizes plasma glutathione, probably by gamma-glutamyl transpeptidase-initiated pathways that account for some, but not all, of the turnover; thus, there is recovery or transport of cysteine moieties. These studies provide an animal model for the human diseases involving glutathione deficiency and are relevant to oxidative phenomena that occur in the newborn.

Identification of a highly reactive threonine residue at the active site of gamma-glutamyl transpeptidase.

gamma-Glutamyl transpeptidase [(5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2], an enzyme of major importance in glutathione metabolism, was inactivated by treating it with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-[3-14C]isoxazoleacetic acid. This selective reagent binds stoichiometrically to the enzyme; more than 90% of the label was bound to its light subunit. Enzymatic digestion of the light subunit gave a 14C-labeled peptide that corresponds to amino acid residues 517-527 of the enzyme and two incomplete digestion products that contain this labeled peptide moiety. The radioactivity associated with this peptide was released with threonine-523 during sequencing by the automated gas-phase Edman method. The light subunit contains 14 other threonine residues and a total of 19 serine residues; these were not labeled. Threonine-523 is situated in the enzyme in an environment that greatly increases its reactivity, indicating that other amino acid residues of the enzyme must also participate in the active-site chemistry of the enzyme.